Human immunodeficiency virus type 1 (HIV-1) infects cells that express the CD4 receptor (De Wolf et al., 1988, AIDS Res Hum Retroviruses 1988; 4:433-440) and, as a result, depletes its host of CD4 lymphocytes (Lang et al., 1989, J Acquir Immune Defic Syndr 1989; 2:63-69). This depletion of CD4 T lymphocytes has been linked to the immunopathogenesis of HIV infection and progression of the disease (Fahey et al., 1990, N Engl J Med 1990; 322:166-172; Masur, 1989, Ann Intern Med 1989; 111:223-231).
A CD4 count of <=200 cells/μl has been included as an AIDS-defining event (Centers for Disease Control. 1992B, Morbid Mortal Weekly Rep 1992; 41(RR-17):1-35), as these measurements are useful predictors for the onset of opportunistic diseases such as Pneumocystis carinii pneumonia (Centers for Disease Control. 1992A, Morbid Mortal Weekly Rep 1992; 41(RR-4):1-11). With the advent of highly active antiretroviral therapy, CD4 T-lymphocyte measurements have been used to monitor immune reconstitution (Autran et al., 1997, Science 1997; 277:112-116).
The current predicate methodology for determining absolute CD4 T-lymphocyte counts is dependent upon immunophenotypic identification of cells with fluorescently labelled monoclonal antibodies directed against the CD4 antigen. Relative percentages of CD4 T cells are determined with a flow cytometer. An absolute CD4 count is derived by multiplying the percentage of lymphocytes that are CD3+ CD4+ by the absolute lymphocyte count determined with a hematology instrument.
However, the overnight shipment of blood may result in increased intrinsic variability in the absolute lymphocyte count depending on the hematology instrument that is used (Koepke and Landay 1989, Clin Immunol Immunopathol 1989; 52:19-2; Paxton et al., 1993, Ann N Y Acad Sci 1993; 677:440-443). Therefore, the absolute CD4 count in overnight samples may have increased variability due solely to the hematological determinants.
Thus, the determination of the “absolute count” of a cell type, i.e., the number of cells in a given volume (concentration), is an important consideration in the general field of cytometry and the field of HIV diagnosis and monitoring in particular.
U.S. Pat. No. 4,110,604 describes a method and apparatus for determining the concentration of particles in a fluid, for example, platelets, through use of a second “reference” particle suspended in the fluid (e.g., red blood cells). The “reference” particle differs in a physical characteristic, for example, electrical impedance, from the particle of interest and is present at a predetermined or determinable concentration or density. The number of red blood cells is counted, as is the number of platelets. Then, by knowing or determining the number of red blood cells in a given unit of volume, an equation can be used to arrive at the number of platelets in the same unit volume. Alternatively, a reference particle could be included in the sample at a known concentration, and then the reference particle is counted along with the platelets. By knowing the concentration of reference particles, it is possible to determine the concentration of platelets.
Such a system as described in U.S. Pat. No. 4,110,604 discriminates between the particles based on their physical characteristics, and is not amenable for use to distinguish between two subpopulations of the same cell type, for example CD3+ CD4+ and CD3+ CD8+ cells. Use of flow cytometry and cell binding agents (capable of binding to cell markers) partially overcomes this problem. Thus, the need for precise and reproducible monitoring of CD4 T-lymphocyte levels in HIV-infected patients has led several companies to develop simpler methods for measuring absolute CD4 and CD8 T-lymphocyte counts (Bene et al., 1998; Denny et al., 1995; Nicholson et al., 1994; O'Gorman, et al., 1997; Paxton, et al., 1995).
European Patent EP 0470810 describes a method for determining the absolute counts of a cell population within a sample by means of flow cytometry. The method makes use of a tube. The tube may comprise a diluent, and the sample is added to the tube containing the diluent. The diluent is said to comprise a solution of isotonic buffer such as phosphate buffered saline, one or more cell markers capable of labelling cells in the population of interest, a fixative such as paraformaldehyde, and a known number of fluorescent microparticles.
The specification of EP 0470810 specifically envisages that the tube may be coated with blocking agents such as bovine serum albumin, caesin or gelatin to prevent adhesion of the components of the diluent to the tube walls, and that these blocking agents may be coated on and dried in the tube using a preservative such as trehalose.
In the method described in EP 0470810, the sample is added to the tube, and the cells are allowed to be labelled with the cell markers. A fluorescence trigger is set to include essentially all the microparticles and cells, and one or more fluorescence gates are set to distinguish between these. The number of cells which meet or exceed the fluorescence trigger are counted, and the number of cells per microparticle for each fluorescence gate is multiplied by the known concentration of microparticles to arrive at the absolute count of cells per unit volume. Thus, knowledge of the number of cells for any population and number of microparticles provides a ratio. Knowing the number of microparticles per unit volume and then multiplying by the first ratio provides the number of cells in a population per unit volume, i.e., the absolute count of the cells.
However, the method described in EP 0470810 makes use of tubes containing a large volume of diluent, which results in certain disadvantages. The diluent present in the tubes described in EP 0470810 necessitates prolonged incubation time and higher amounts of the cell-binding agents (and thus higher cost). Furthermore, the presence of the diluent impedes efficient lysing of the erythrocytes. Erythrocyte lysing is a procedure recommended by the Centres for Disease Control and Prevention (CDC), The European Working Group on Clinical Cell Analysis (EWGCCA) and The British Committee for Standards in Haematology (BCSH) for enumeration of CC3-CD4 positive cells.
The percentage of CD4+ cells out of the total lymphocyte population cannot be determined using the method described in EP 0470810 since there is no marker for total lymphocytes included. This is crucial in paediatric patients where a CD4 count is not sufficient for monitoring disease progression. Children under the age of 5 have higher levels of CD4 positive lymphocytes than adults and thus a percentage of CD4+ cells out of the total lymphocytes is necessary for determining whether treatment should be initiated. Close monitoring of HIV infection is of particular importance in children, since the infection can actually be cured in very young patients.
Finally, the large volume of the diluent means that the tubes described in EP 0470810 are not easily adapted for manufacture as a disposable component, leading to a higher price.
Disposable containers for absolute cell counting are known in the art. Such containers comprise dispensed portions or aliquots containing a known, fixed number of microparticles per tube. Knowledge of the number of microparticles and, crucially, maintenance of this number within the tube during handling (e.g., prior to and during addition of the sample), is essential to the accuracy of the counts obtained.
It is known for example, to employ disposable containers containing a pre-determined number of microparticles. For example, a commercially available counting system comprises BD TruCOUNT Tubes (Catalog No. 340334, BD Biosciences San Jose, Calif.). TruCOUNT absolute-count tubes contain a lyophilised pellet that dissolves during sample preparation, releasing a known number of fluorescent beads. The tube comprises a stainless steel retainer in the form of a grid which is positioned near the closed end of the tube and above the lyophilised pellet. The stainless steel retainer prevents the lyophilised pellet from falling out of the container during routine handling (such as for example, inversion or shaking of the tube), and accordingly maintains the fixed predetermined number of microparticles in the tube.
Nevertheless, problems remain with such embodiments. These generally arise from the fact that the lyophilisation results in a pellet, which is “fluffy” and easily breaks up from handling. Specifically, it is crucial to avoid disturbing the lyophilised pellet during sample handling and addition. Thus, the operating instructions for the TruCOUNT tubes specifically caution against disturbing the steel retainer and the pellet containing the beads, and advise pipetting above the stainless steel retainer. Furthermore, depending on the pitch of the stainless steel grid, it may not be totally effective in preventing portions of the lyophilised pellet from being detached and falling out of the tube. In order to minimise disturbance to the lyophilised pellet and grid, the TruCOUNT tubes have to be packaged in a protective pouch in a controlled atmosphere. Once the protecting pouch has been opened, the pellet will absorb moisture and consequently shrink. When this happens, the pellet is at risk of falling through the grid. Thus, when any of the tube, the pellet or grid is disturbed, or portions are lost, the absolute count obtained is potentially subject to error. The operating instructions for the TruCOUNT tubes state that the tubes should be discarded if the pellet has been disturbed in any manner.
Other problems arise from the nature of the beads employed in the TruCOUNT tubes. These give a very small forward scatter signal on the flow cytometer, and therefore the beads are seen in the same area as debris from lysed erythrocytes or unlysed erythrocytes. This means that it is not possible to use a trigger on the forward scatter parameter since this would either include both beads and debris/erythrocytes or exclude both. Instead a fluorescence trigger has to be used, which is a disadvantage, as some users prefer to use a scatter trigger instead of a fluorescence trigger.
Furthermore, methods of absolute counting which employ microbeads need to be carefully optimised to maintain the precision and consistency (i.e., count to count variability) of counts. One primary problem is that not all beads in the mixed sample may be counted, i.e., the actual number of beads counted with the flow cytometer is lower than expected from the predetermined number present in the tube. This arises due to the tendency of microparticles to adhere to one another to form doublets, triplets, quadruplets, etc. This is a particular problem with the beads used in the TruCOUNT tubes described above, in which (depending on batch) there is usually 5-10% multiple beads in a tube. Although this can be accounted for in the bead value quoted, it may give rise to discrepancies in the absolute counts.
In addition to adhering to one another, microparticles also tend to adhere to the surface of the container. Finally, some particles are not registered by the flow cytometer due to dead time in the sample acquisition. These factors compound to cause a discrepancy between the number of beads applied to the flow cytometer and the number of beads registered by the detectors of the flow cytometer in prior art methods.
A further problem is inter-count variability, i.e, the consistency of counts obtained through repeated processing. In other words, it is of the outmost importance that the variation in count between identical samples is low, i.e. one obtains a similar count every time a particular sample is measured. Here, the primary cause of the non-reproducibility appears to be microparticles adhering to the walls of the container in variable numbers.
The primary cause of this is the “stickiness” of the microparticles, i.e., the tendency of the microparticles to adhere to other components. This appears to be dependent on the nature of the material from which the microparticles are made, and the conditions in the environment in which they are counted. Variables such as pH, ionic strength, hydrophobicity and temperature of the sample medium can and do cause microparticles to have increased adhesiveness. Coating the walls of the container with for example BSA can reduce but not completely eliminate the problem. Multiplying the count obtained with a correction factor to account for the “lost” beads may also help reduce the discrepancy.
Nevertheless, it is clear that there are problems in the prior art, which have the potential to reduce the precision and accuracy of the absolute counts.